Single-molecule counting of circular RNAs using phage nanoparticles as surrogates Abstract Circular RNA (circRNA) have been considered as promising breast cancer biomarkers, but the current qRT-PCR and digital PCR techniques for quantifying them have some inherent limitations. T7 phage, a human-safe virus nanoparticle specifically infecting bacteria, can be plated to infect bacteria in a plaque-forming assay to form millimeter-scale plaques in a one to one format and at a single-particle resolution within 3 hours. Inspired from this, we propose to develop a phage plaque counting (PPC) strategy that hires bioengineered fluorescent T7 phage as a surrogate to establish a one-to-one correspondence among target circRNA, phage nanoparticles, and eye-visible phage-developed plaques, enabling us to simultaneously quantify multiple circRNA biomarkers in a single test by simply counting the corresponding plaques. Briefly, a fluorescent phage probe with an oligonucleotide (ONT-1) capable of capturing one unique segment of a target circRNA, and a magnetic microparticle (MMP) probe with an ONT-2 capable of capturing another unique segment of the same target, co- capture the target to form a sandwich complex where phages and target molecules are equimolar. Then the phages are released and plated to develop fluorescent plaques in a one to one format, thus counting the plaques at the single-particle level leads to visualized quantification of the target circRNA at a single-molecule level. A fluorescent protein on the capsid makes the corresponding plaque fluoresce a unique color and enables the simultaneous single-particle quantification of fluorescent plaques (in the same Petri dish) with each color coding one target (i.e., by displaying green and red fluorescent protein for two corresponding targets). We hypothesize that our PPC strategy with optimized conditions can simultaneously quantify a panel of two circRNAs as breast cancer biomarkers in human serum with high sensitivity, specificity, and reproducibility. Aim 1: Establish and optimize PPC strategy for quantifying single and multiple circRNA breast cancer biomarkers. We will produce and purify the target circRNAs (circ_0001785 and circ_100219) by the overexpression method and use the PPC method to quantify them with a series of dilutions in water. We will optimize the PPC and identify its detection limit. Aim 2: Validate the PPC strategy for simultaneously quantifying multiple circRNA biomarkers in breast cancer cells, tissues, and human serum. We will first use MMPs that can capture the target circRNAs to magnetically remove the pre-existing target circRNAs from the commercial human serum, which is then used to make serum samples with known concentrations of target circRNAs. Then we will employ and optimize the PPC to quantify the target circRNAs in the serum. We will also isolate total RNAs from breast cancer cells and in vitro breast tumor tissues to form aqueous RNA solutions by a commercial RNA isolation kit. We will then use the PPC to quantify the target circRNAs in the resultant RNA solutions and the commercial total RNA solutions isolated from tumor tissues of breast cancer patients. This project will lead to a new visualized single-molecule technique for quantifying circRNA biomarkers for breast cancer diagnosis.